Quantitative Detection of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> by Real-Time PCR in Raw Milk

Clostridium perfringens causes a broad spectrum of diseases in both humans and animals and is an important cause of foodborne illness. We developed and tested a real-time (Q-)PCR assay for the species-specific detection of C. perfringens that targets the phospholipase C (plc) gene and includes an internal amplification control (IAC), making it possible to identify false-negative results, which are common due to the high level of PCR inhibition by food compounds. The CPplc-IAC real-time PCR (RTi-PCR) assay was 100% selective, as shows with 36 Clostridium strains and 85 non-Clostridium strains, with an analytical sensitivity of 1 genome equivalent (GE) in 23% of the reactions and 10 GE in 100% of the reactions. The quantification was linear (R ² = 0.9990) over a 7-log dynamic range, down to 10 GE, with a PCR efficiency E = 0.841. The applicability of this RTi-PCR assay was assessed in milk samples. The assay detected as few as 300 spores in 25 mL of artificially contaminated raw sheep milk with 78% probability and 30 spores in 25 mL with 50% probability. It also has accuracy of 83.03 to 151.18%, as shown by an evaluation of the correspondence between RTi-PCR assay results and the number of spores per milliliter determinated by standard plating. This RTi-PCR method was effective for the detection and quantification of C. perfringens in milk having an important applicability in the control of this pathogen in the dairy food industry.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

A Pilot Study for Identification of <Emphasis Type="Italic">Salmonella</Emphasis> in Food Processing Plants by Real-Time PCR Screening

Salmonella remains a major public health concern worldwide. Microbiological methods are the gold standard for Salmonella detection. These methods are highly specific, but their sensitivity is variable. Moreover, they are lengthy, labour intensive and not always consistent with the speed of food manufacturing processes. Thus, in the food industry, there is the need for more rapid, sensitive and accurate detection methods. The purpose of this study is to describe a Salmonella-monitoring scheme in different food processing plants based on a screening approach by a commercial real-time polymerase chain reaction (PCR) kit and subsequent confirmation of positive molecular results by the reference microbiological method. This scheme was tested on a total of 4,693 samples, 90 of which were positive with the real-time PCR screening; 52 of the positive samples were eventually confirmed by the microbiological method. The real-time PCR kit was tested in comparison to the microbiological method in order to evaluate its performances and drawbacks. The comparison between cycle threshold (Ct) values of real-time PCR and the microbiological results (Wilcoxon rank sum test) showed a statistically significant difference between the Ct values of bacteriological positive and bacteriological negative samples (p value, <0.05). Furthermore, receiver operating characteristic curve analysis was used to identify the Ct value ensuring the lowest level of misclassification between Salmonella-positive and negative samples. The present study confirms that the real-time PCR kit tested could be used as a screening tool, leading to a rapid and sensitive identification of Salmonella and confining bacteriological confirmation to samples previously identified as positive.     

Estrogenic effects of various extracts from <Emphasis Type="Italic">Chamdanggui</Emphasis> (<Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai) and <Emphasis Type="Italic">sogdan</Emphasis> (<Emphasis Type="Italic">Phlomis umbrosa</Emphasis> Turcz)

The various extracts from chamdanggui (Angelica gigas Nakai) and sogdan (Phlomis umbrosa Turcz) were evaluated for estrogenic activity and characterized according to HPLC profile. Chamdanggui and sogdan were individually extracted with 4 solvents (hot water, 70% ethanol, n-butanol, and dichloromethane) of differing polarities. Estrogenic activity was determined by E-screen using an estrogen-dependent MCF-7 BUS cell. Although almost all extracts showed estrogenic effects in a concentrationdependent manner, the hot water extract from chamdanggui (250 μg/mL) had the higher effect (138%). Among 90 fractions using HPLC separation of the hot water extract from chamdanggui, fraction 21 and 28 produced the highest estrogenic effects of 178 and 163% at 10 μg/mL, respectively. The results imply that the hot water extract from chamdanggui could be useful as an alternative hormone replacement therapy.     

Thermal inactivation kinetics of quality-related enzymes in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis><Emphasis Type="Bold">)</Emphasis>

Thermal inactivation of quality-related enzymes in both cauliflower crude enzyme extracts and fresh tissue samples was studied in temperature range 50–100 °C. For crude enzyme extracts, several parameters, reaction rate constants (k) and activation energy (E _a) as well as decimal reduction time (D) and (z) values, were used to characterize the thermal stability. The rates of inactivation were found to follow first-order inactivation kinetics. Activation energies varied between 101.18 and 208.42 kJ mol⁻¹ with z values of 10.59–24.09 °C. The examined kinetics indicated that lipoxygenase was the most heat resistant followed by peroxidase, polyphenol oxidase, pectin methyl esterase and ascorbic acid oxidase. Furthermore, the obtained results from the blanched fresh tissues indicated that inactivation of lipoxygenase secured disappearing of any other enzyme activities. Therefore, this study recommends using lipoxygenase as an indicator enzyme to optimize the thermal treatments of cauliflower products.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.     

Molecular diagnostics of Lemon Myrtle (<Emphasis Type="Italic">Backhousia citriodora</Emphasis> versus <Emphasis Type="Italic">Leptospermum citratum</Emphasis>)

‘Lemon Myrtle’ is becoming increasingly popular in Europe both for use in cuisine and phytotherapy. However, this common name covers two completely different species, Backhousia citriodora F. Muell. and Leptospermum citratum Challinor, Cheel & A.R.Penfold. These species differ with respect to secondary compounds and even can cause, if mixed up and applied in high dose, toxic effects. We describe how the two species can be discriminated microscopically making use of differences in the morphology of leaf pavement cells and the relative size of palisade parenchyma. Based on the large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL) as molecular marker, the phylogenetic position of the two species within the Myrtaceae could be clarified. This sequence information was used to develop a simple assay to discriminate the two species even in dried and highly fragmented mixtures as typically occurring in commercial samples. This assay utilises the occurrence of single-nucleotide exchanges between those species that produce different fragments when the rbcL amplificates are restricted with Sac II.     

Pressurized acidified water extraction of black carrot [<Emphasis Type="Italic">Daucus carota</Emphasis> ssp. <Emphasis Type="Italic">sativus</Emphasis> var. <Emphasis Type="Italic">atrorubens</Emphasis> Alef.] anthocyanins

The anthocyanins present in black carrot were extracted with pressurized water acidified with sulfuric, citric and lactic acids. Anthocyanin degradation became significant above 100 °C and there was no improvement when extraction pressure was increased to 100 bar. Therefore, the extraction from black carrot was carried out at temperatures 50, 75 and 100 °C under 50 bar pressure. The extraction efficiencies in terms of acylated and non-acylated anthocyanins were comparable for all three acids used to acidify water at 50 °C, while similar results were observed at 75 °C for both citric and lactic acids. Water acidified with lactic acid showed significantly higher extraction efficiency at 100 °C compared to water acidified with sulfuric and citric acids. Highest degree of polymerization together with increasing degree of browning was observed within extracts when sulfuric acid was used. On the other hand, when organic acids were used to acidify water, a higher extraction efficiency of anthocyanins, accompanied with a relatively low polymerization and browning was observed, with lactic acid giving the best results.     

Rapid Detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> in Shellfish by Real-Time Recombinase Polymerase Amplification

Vibrio parahaemolyticus (V. parahaemolyticus) is a zoonotic pathogen generally found in seafood. To detect the foodborne pathogen rapidly and accurately for food safety measures, we developed a real-time recombinase polymerase amplification (RPA) method. An evaluation of the specificity and sensitivity of the method is discussed here. A set of primers and probe was specially designed to target the tlh gene, which is usually regarded as a marker of total V. parahaemolyticus strains. During the reaction, target DNA was amplified and tagged with specific fluorophore within 10 min and at an incubation temperature of 40 °C. In addition to fast amplification and low temperature, the fluorescence signal was synchronized with the amplification of products for the generation of real-time data. The detection limit of this assay was 0.4 pg/μL of DNA, which is comparable to assays that use the bacterial culture as template, 4?×?10³ cfu/mL. The real-time RPA method had a stable performance when testing the spiking shellfish samples at the same level of contamination by the pathogen in different kinds of shellfish. Thus, the real-time RPA method shows great potential for on-site detection of V. parahaemolyticus, especially in low-resource settings.     

Effect of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Bauer and <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> BB12 on proteolytic changes in dry-cured loins

The effect of the potentially probiotic bacteria strain of Lactobacillus acidophilus Bauer and probiotic bacteria Bifidobacterium animalis ssp. lactis BB12 on proteolytic changes of proteins in dry-cured loins during fermentation and cold storage was studied. Results of the conducted tests demonstrated that the use of probiotic bacteria for the production of dry-cured meats impacts the generation of products of protein proteolysis with high antioxidant activity. The highest antioxidant activity of peptides after fermentation and cold storage was observed in the loin with the strain B. animalis ssp. lactis BB12 and the loin with the mixture of strains L. acidophilus Bauer and B. animalis ssp. lactis BB12. Qualitative analysis of peptides demonstrated that peptides with weight below 3.5 kDa are characterized by the highest capacity of quenching the ABTS cation radical, including the peptides in loins with the strain B. animalis ssp. lactis BB12.     

In vivo study of antiallergenicity of ethanol extracts from <Emphasis Type="Italic">Sargassum tenerrimum</Emphasis>, <Emphasis Type="Italic">Sargassum cervicorne</Emphasis> and <Emphasis Type="Italic">Sargassum graminifolium turn</Emphasis>

Food allergy has becoming the serious threat in the world for which the search of an effective anti-allergic drug is the demand of time. Keeping in view of the potentiality of seaweeds, the ethanol extracts from Sargassum tenerrimum (ST), Sargassum cervicorne (SC), and Sargassum graminifolium turn (SG) have been studied in vivo for its antiallergenicity through passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) in female BALB/c mice. Intraperitoneal administration of these ethanol extracts inhibit mouse PCA and ACA in a dose-dependent manner using ovalbumin (OVA) and shrimp allergen as triggering agents to induce allergenicity over mice. The extract of ST containing phlorotannin has been found most active over the suppression of PCA triggered by OVA and shrimp with IC₅₀ values of 25.64 and 40.98 mg/kg, respectively and an efficacy comparable to that of an anti-allergic drug disodiumcromoglycate. Similarly, ST inhibits ACA triggered by ova and shrimp allergen in the mouse, with 50% suppression at 25.5 and 43.53 mg/kg, respectively. The results presented here show that these extracts are active on the studied models among which ethanol extract of ST was the most potent, leading toward the promising development of a new class of anti-allergic drugs.     

Spray Chilling Microencapsulation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> and Its Use in the Preparation of Savory Probiotic Cereal Bars

The use of probiotic microorganisms has been limited by the difficulty of maintaining their viability during processing and throughout the product’s shelf life. This study evaluated the viability of microencapsulating Lactobacillus acidophilus (LA) and Bifidobacterium animalis subsp. lactis (BL) using the spray chilling technique to add them to savory cereal bars. The results showed that spray chilling generated a powder that was composed of smooth and continuous spheres with low moisture content and low water activity. The microencapsulated microorganisms exhibited a storage viability at least of 90 days as microparticles and in savory cereal bars, and their counts were superior to those resulting from other methods of adding activated and lyophilized probiotics to savory cereal bars. Thus, microparticles prepared by spray chilling are good vehicles for incorporating probiotics into cereal bars and have the potential to release the probiotics in the consumers’ intestines by means of fat digestion. Savory cereal bars that did and did not contain probiotics exhibited no differences in sensorial acceptance or commercial potential.     

Irradiation Sensitivity of Planktonic and Biofilm-associated <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">L. innocua</Emphasis> as Influenced by Temperature of Biofilm Formation

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D ₁₀ value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D ₁₀ values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D ₁₀ values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.     

Effect of heat-treated <Emphasis Type="Italic">Nuruk</Emphasis> on the quality characteristics of aged <Emphasis Type="Italic">Yakju</Emphasis>

Long-term aging of Yakju, a traditional Korean liquor made of rice and Nuruk (a fermentation agent), causes browning and odor and flavor development. This study investigated the effects of heat-treated Nuruk (50–80 °C, 30 min) on Yakju quality. The saccharogenic powers and glucoamylase, α-amylase, and carboxypeptidase activities were similar in non-heat-treated Nuruk and that treated at 50 °C. However, acidic protease and alcohol dehydrogenase decreased above 50 °C. The content of nitrogen-containing compounds was inversely proportional to the heat-treatment temperature. Compounds that cause off-flavors decreased at 50–60 °C, but increased at 70–80 °C, whereas compounds that provide fragrance increased at 50–60 °C. Sensory evaluation indicated that bad taste attributes were higher in Yakju produced using non-heat-treated Nuruk. Therefore, heat treatment of Nuruk at 50 °C can be adopted as a method for improving Yakju quality, as enzymatic activities that affect color, aroma, and taste are regulated.     

Differences of Bactericidal Efficacy on <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>, and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> of Slightly and Strongly Acidic Electrolyzed Water

In the present study, the disinfection efficacy of slightly acidic electrolyzed water (SAEW) and strongly acidic electrolyzed water (AEW) was tested on three bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis and the disinfection mechanism was discussed. The results showed that SAEW had a stronger antibacterial efficacy against these tested bacteria in comparison with AEW. The results also showed that both SAEW and AEW treatments could damage the cell membrane, which was demonstrated microcosmically by scanning electron microscopy (SEM), thus causing leakages of protein, DNA, RNA, and ATP, resulting in the death of microbes. Moreover, AEW treatment could not cause the degradations of DNA and RNA, and nucleic acids including DNA and RNA are not the target point of its bactericidal efficacy. However, SAEW could maybe cause the degradation of RNA, and RNA may be the target in its antibacterial activity. We suggested that the differences in antibacterial efficacy between SAEW and AEW could be explained by the different impacts on RNA of tested strains.     

Prevalence,genetic diversity,and antibiotic susceptibility of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from <Emphasis Type="Italic">Sunshik</Emphasis>, its ingredients and soils

The prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter species (Enterobacter sakazakii) isolated from sunshik products, its ingredients, and root vegetable farm’s soils were investigated to analyze main reservoirs and contaminated sources of Cronobacter spp. Cronobacter spp. was isolated from 9 of 15 sunshik products, 26 of 72 its ingredients, and 2 of 39 soils. The root vegetables such as sweet potato and carrot showed higher contamination rate (70%) than the other sunshik ingredients. All isolates showed 929 bp band amplified from 16S rRNA and resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin. All isolates showed diverse random-amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) band patterns. However, 3 cases of RAPD banding patterns between clinical strains and isolates from sunshik products and root vegetables (yam, carrot) were related with similarities level of 80%. These studies indicated that root vegetables can be an important contamination source of Cronobacter spp. in sunshik products. Thus, the preparation of root vegetables for manufacturing sunshik products used as a weaning diet was handled with more care than the other sunshik ingredients.     

Tyramine reduction by tyrosine decarboxylase inhibitor in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> for tyramine controlled <Emphasis Type="Italic">cheonggukjang</Emphasis>

This study was carried out to find a method to control tyrosine decarboxylase activity (TDC) of a strain of Enterococcus faecium capable of producing high levels of tyramine. To select a TDC inhibitor, enzyme assay was first performed using purified TDC enzyme and 0.1% of TDC inhibiting chemicals. When 0.23% of nicotinic acid was added, tyramine content (363 ug/mL) was lower than that of the control group (873 ug/mL). At the same time, bacterial growth was decreased 1 log cycle from 8.62 to 7.56 log CFU/mL. TDC expression level in E. faecium was measured by using RT-qPCR. Lower expression level (below 0.7) was observed after the addition of 0.23% nicotinic acid (in vitro). When cheonggukjang was manufactured with addition of nicotinic acid, tyramine contents were decreased from 698.67 to 117.27 mg/kg when the concentration of nicotinic acid added was increased from 0.10 to 0.30%. These results suggest that nicotinic acid could be used as an agent (TDC inhibitor) to reduce tyramine content in cheonggukjang.